Isotopic analyses are useful for diagnosis of a disease in a medical application, in which metabolic functions of a living body can be determined by measuring a change in the concentration or concentration ratio of an isotope after administration of a drug containing the isotope. In the other fields, the isotopic analyses are used for studies of the photosynthesis and metabolism of plants, and for ecological tracing in a geochemical application.
It is generally known that gastric ulcer and gastritis are caused by bacteria called helicobacter pylori (HP) as well as by a stress. If the HP is present in the stomach of a patient, an antibiotic or the like should be administered to the patient for bacteria removal treatment. Therefore, it is indispensable to check if the patient has the HP. The HP has a strong urease activity for decomposing urea into carbon dioxide and ammonia.
Carbon has isotopes having mass numbers of 12, 13 and 14, among which .sup.13 C having a mass number of 13 is easy to handle because of its non-radioactivity and stability.
If the concentration of .sup.13 CO.sub.2 (a final metabolic product) or the concentration ratio of .sup.13 CO.sub.2 to .sup.12 CO.sub.2 in breath of a patient is successfully measured after urea labeled with the isotope .sup.13 C is administered to the patient, the presence of the HP can be confirmed.
However, the concentration ratio of .sup.13 CO.sub.2 to .sup.12 CO.sub.2 in naturally occurring carbon dioxide is 1:100. Therefore, it is difficult to determine the concentration ratio in the breath of the patient with high accuracy.
There have been known methods for determining the concentration ratio of .sup.13 CO.sub.2 to .sup.12 CO.sub.2 by means of infrared spectroscopy (see JPB 61(1986)-42219 and JPB 61(1986)-42220).
In the method disclosed in JPB 61(1986)-42220, two cells respectively having a long path and a short path are provided, the path lengths of which are adjusted such that the light absorption by .sup.13 CO.sub.2 in one cell is equal to the light absorption by .sup.12 CO.sub.2 in the other cell. Light beams transmitted through the two cells are lead to spectrometric means, in which the light intensities are measured at wavelengths each providing the maximum sensitivity. In accordance with this method, the light absorption ratio can be adjusted to "1" for the concentration ratio of .sup.13 CO.sub.2 to .sup.12 CO.sub.2 in naturally occurring carbon dioxide. If the concentration ratio is changed, the light absorption ratio also changes by the amount of a change in the concentration ratio. Thus, the change in the concentration ratio can be determined by measuring a change in the light absorption ratio.
(A) However, the method for determining the concentration ratio according to the aforesaid document suffers from the following drawbacks.
Calibration curves for determining the concentrations of .sup.12 CO.sub.2 should be prepared by using gaseous samples each having a known .sup.12 CO.sub.2 concentration.
To prepare the calibration curve for the .sup.12 CO.sub.2 concentration, the .sup.12 CO.sub.2 absorbances are measured for different .sup.12 CO.sub.2 concentrations. The .sup.12 CO.sub.2 concentrations and the .sup.12 CO.sub.2 absorbances are plotted as abscissa and ordinate, respectively, and the calibration curve is determined by the method of least squares.
The calibration curve for the .sup.13 CO.sub.2 concentration is prepared in the same manner as described above.
For determination of the concentrations by means of infrared spectroscopy, the preparation of the calibration curves is based on an assumption that the relation between the concentration and the absorbance conforms to the Lambert-Beer Law. However, the Lambert-Beer Law itself is an approximate expression. The actual relation between the concentration and the absorbance does not always conform to the Lambert-Beer Law. Therefore, all the plotted data do not perfectly fit to the calibration curve.
FIG. 1 is a graphical representation in which concentration ratios of .sup.13 CO.sub.2 to .sup.12 CO.sub.2 are plotted with respect to .sup.12 CO.sub.2 concentrations, the .sup.12 CO.sub.2 concentrations and the .sup.13 CO.sub.2 concentrations having been determined by using calibration curves prepared on the basis of measurements of the absorbances of gaseous samples having the same concentration ratio (.sup.13 CO.sub.2 concentration/.sup.12 CO.sub.2 concentration=1.077%) but different .sup.12 CO.sub.2 concentrations.
As shown in FIG. 1, the concentration ratios determined for different .sup.12 CO.sub.2 concentrations deviate from the actual concentration ratio (1.077%), and form an undulatory curve when plotted.
Although the cause of the deviation has not been elucidated yet, the deviation supposedly results from changes in the spectroscopic characteristics such as reflectance, refractive index and stray light in dependence on the .sup.12 CO.sub.2 concentration and from the error characteristics of the least square method employed for the preparation of the calibration curves.
If the concentration of a component gas is determined without correction of the characteristics associated with the deviation, a critical error may result.
(B) A variety of experiments have revealed that, where the infrared spectrometry is employed to measure the concentration of .sup.13 CO.sub.2 or the concentration ratio of .sup.13 CO.sub.2 to .sup.12 CO.sub.2 (hereinafter referred to as ".sup.13 CO.sub.2 concentration ratio"), measurement results differ from the actual .sup.13 CO.sub.2 concentration or .sup.13 CO.sub.2 concentration ratio depending on the concentration of oxygen contained in a gaseous sample.
FIG. 2 is a graphical representation in which .sup.13 CO.sub.2 concentration ratios are plotted with respect to oxygen contents, the .sup.13 CO.sub.2 concentration ratios having been determined by measuring gaseous samples containing .sup.13 CO.sub.2 diluted with oxygen and nitrogen and having the same .sup.13 CO.sub.2 concentration but different oxygen concentrations. The determined .sup.13 CO.sub.2 concentration ratios are normalized on the basis of a .sup.13 CO.sub.2 concentration ratio for an oxygen content of 0%.
As shown in FIG. 2, the .sup.13 CO.sub.2 concentration ratio is not constant but varies depending on the oxygen concentration.
If the .sup.13 CO.sub.2 concentration or the .sup.13 CO.sub.2 concentration ratio of a gaseous sample containing oxygen is measured in ignorance of this fact, it is obvious that a measurement differs from an actual value.
FIG. 3 is a graphical representation illustrating the result of measurement in which gaseous samples having different .sup.13 CO.sub.2 concentration ratios and containing no oxygen were measured. In FIG. 3, the actual .sup.13 CO.sub.2 concentration ratios and the measured .sup.13 CO.sub.2 concentration ratios are plotted as abscissa and ordinate, respectively. The .sup.13 CO.sub.2 concentration ratios are normalized on the basis of the minimum .sup.13 CO.sub.2 concentration ratio.
FIG. 4 is a graphical representation illustrating the result of measurement in which gaseous samples having different .sup.13 CO.sub.2 concentration ratios and containing various concentration of oxygen (up to 90%) were measured. In FIG. 4, the actual .sup.13 CO.sub.2 concentration ratios and the measured .sup.13 CO.sub.2 concentration ratios are plotted as abscissa and ordinate, respectively. The .sup.13 CO.sub.2 concentration ratios are normalized on the basis of the minimum .sup.13 CO.sub.2 concentration ratio.
A comparison between the results shown in FIGS. 3 and 4 indicates that the relationship between the actual .sup.13 CO.sub.2 concentration ratio and the measured .sup.13 CO.sub.2 concentration ratio in FIG. 3 is about 1:1 (or the scope of the fitting curve in FIG. 3 is about 1) while the relationship between the actual .sup.13 CO.sub.2 concentration ratio and the measured .sup.13 CO.sub.2 concentration ratio in FIG. 4 is about 1:0.3 (or the scope of the linear fitting curve in FIG. 4 is about 0.3).
Thus, the measurement of the .sup.13 CO.sub.2 concentration or the .sup.13 CO.sub.2 concentration ratio is influenced by the concentration of oxygen contained in a gaseous sample, the cause of which has not been elucidated yet.
If the concentration or concentration ratio of a component gas is determined without performing a correction in consideration of the oxygen concentration, it is predicted that a critical error may result.
(C) Since the concentration of CO.sub.2, particularly, the concentration of .sup.13 CO.sub.2 is extremely low, highly sensitive measurement is required. When the sensitivity of measurement is increased, a measured light intensity is responsive to changes in parameters of the measurement system, e.g., the light intensity of a light source, the temperature of a sample gas, the temperature of a cell to which the gas is introduced, the sensitivity of a photodetector and the like. Thus, the measured value may have an error caused by factors not related to the sample gas.
To solve this problem, the measurement is started after the measurement system is stabilized in a time-consuming manner. This reduces the operation efficiency and makes it impossible to meet a user demand to measure a large amount of samples in a short time.
For measurement of one breath sample, the .sup.12 CO.sub.2 absorbance is measured and the .sup.12 CO.sub.2 concentration is determined on the basis of a calibration curve for .sup.12 CO.sub.2. The .sup.13 CO.sub.2 absorbance is measured and the .sup.13 CO.sub.2 concentration is calculated on the basis of a calibration curve for .sup.13 CO.sub.2, as well. The measurement of another breath sample is carried out in the same manner.
If the CO.sub.2 concentrations of the aforesaid two breath samples are at substantially the same level, the ranges of the calibration curves for .sup.12 CO.sub.2 and .sup.13 CO.sub.2 to be used for the concentration determination can be limited. Thus, the measurement accuracy can be increased by using limited ranges of the calibration curves.
(D) In a conventional infrared spectrometric method as described above, a bag containing a gaseous sample is connected to a predetermined pipe of a spectrometric apparatus, and the gaseous sample is introduced into a cell through the pipe by manually compressing the bag.
However, even small turbulence may drastically reduce the measurement accuracy because the absorbance of .sup.13 CO.sub.2 present in a trace amount is measured in the isotopic gas analysis. The gaseous sample cannot be passed through the cell at a constant flow rate by the manual compression of the bag. This generates a nonuniform flow of the gaseous sample in the cell and causes the gaseous sample to have a local temperature change and an incidental concentration change, thereby fluctuating a light detection signal.
The flow rate of the gaseous sample may be controlled to be constant by using a pump and a flow meter in combination. However, the accuracy of the flow control cannot be ensured, because the volume of the bag containing the gaseous sample is small and the flow rate is low. Alternatively, an apparatus called mass flow meter for electronic flow control may be employed as flow control means. This improves the accuracy of the flow rate control, but results in a complicated apparatus and an increased cost.
(E) In the method disclosed in JPB 61(1986)-42220, the length of the cell is reduced and, therefore, a cell-absent space is filled with air. The air space hinders highly accurate measurement. If the lengths of paths between the light source and the cell and between the cell and the photoreceptor are increased, highly accurate measurement may be hindered.
More specifically, since the absorbance of .sup.13 CO.sub.2 present in a trace amount is measured in the isotopic gas measurement, even a small external disturbance reduces the measurement accuracy. A few percentage of .sup.12 CO.sub.2 and a trace amount of .sup.13 CO.sub.2 are present in the aforesaid air space and spaces between the light source and the cell and between the cell and the photoreceptor. A .sup.13 CO.sub.2 spectrum partially overlaps a .sup.12 CO.sub.2 spectrum and, if a filter is used, the band-pass width thereof influences the measurement. Therefore, the presence of .sup.12 CO.sub.2 indirectly influences the measurement of the .sup.13 CO.sub.2 absorbance, and the trace amount of .sup.13 CO.sub.2 directly influences the measurement of the .sup.13 CO.sub.2 absorbance.
To eliminate the influence of CO.sub.2 present in a light path, an apparatus (see JPB 3(1991)-31218) has been proposed in which a light source, a sample cell, a reference cell, a interference filter, a detection element and like elements are accommodated in a sealed case which is connected to a column filled with a CO.sub.2 absorbent through a tube and a circulation pump for circulating air within the sealed case and the column to remove CO.sub.2 from the air in the sealed case.
The apparatus disclosed in this document can remove CO.sub.2 which may adversely affect the measurement, but requires the column filled with the CO.sub.2 absorbent, the tube and a large sealed case for accommodating the respective elements, resulting in a large-scale construction. In addition, the fabrication of the apparatus requires a laborious process such as for sealing the large case.
Further, a nonuniform flow of the air within the sealed case causes a local temperature change and an incidental concentration change, thereby causing a light detection signal to be fluctuated.
(F) In the infrared spectroscopic measurement, breath is sampled in breath sampling bags before and after a diagnostic drug is administered to a living body, and the breath samples in the breath sampling bags are respectively measured for determination of the .sup.13 CO.sub.2 concentration or the .sup.13 CO.sub.2 concentration ratio.
The measurement of such breath samples is typically performed in a professional manner in a measurement organization, which manipulates a large amount of samples in a short time. Therefore, breath samples obtained before and after the drug administration are often mistakenly manipulated.
More specifically, breath samples obtained from one patient before and after the drug administration are mistaken for those obtained from another patient, or a breath sample obtained before the drug administration is mistaken for that obtained after the drug administration.
Such mistakes lead to erroneous measurement results and, therefore, should be assuredly prevented.
Further, if a breath sample includes a gas remaining in the oral cavity of a patient, the measurement accuracy is reduced. To reduce a measurement error, breath from the lung of the patient should be sampled.
Still further, since moisture in a breath sample adversely affects the optical measurement, the moisture should be removed from the breath sample. Furthermore, a consideration should be given to the breath sampling bag to prevent the breath sample from escaping from the bag.
It is an object of the present invention to provide a method for spectrometrically measuring an isotopic gas, which is employed to precisely determine the concentration or concentration ratio of a component gas in a gaseous test sample containing a plurality of component gases by way of spectrometry when the gaseous test sample is introduced into a cell.
It is another object of the present invention to provide a method for spectrometrically measuring an isotopic gas, which is employed to precisely determine the concentration of a component gas in a gaseous test sample containing a plurality of component gases by way of spectrometry by using a limited range of a calibration curve when the gaseous test sample is introduced into a cell.
It is further another object of the present invention to provide a method for spectrometrically measuring an isotopic gas, which is employed to precisely determine the concentration or concentration ratio of .sup.13 CO.sub.2 contained in a gaseous test sample by way of spectrometry in consideration of the concentration of oxygen when the gaseous test sample into a cell.
It is still another object of the present invention to provide a method for spectrometrically measuring an isotopic gas, which is employed to precisely determine the concentration or concentration ratio of a component gas in a gaseous test sample containing a plurality of component gases by way of spectrometry in such a manner that time-related influences on a measurement system can be minimized when the gaseous test sample is introduced into a cell.
It is yet another object of the present invention to provide an apparatus for spectrometrically measuring an isotopic gas, which has a simple construction and is capable of introducing a gaseous test sample containing a plurality of component gases at a constant flow rate for spectrometry.
It is still another object of the present invention to provide a breath sampling bag, which is given a consideration to assuredly prevent a breath sample from being mistakenly manipulated.
It is yet another object of the present invention to provide a breath sampling bag, which prevents the sampling of air present in the oral cavity of a patient but allows the sampling of breath from the lung of the patient.
It is still another object of the present invention to provide a breath sampling bag, which is capable of removing moisture from breath blown therein.
It is yet another object of the present invention to provide a breath sampling bag, which has a construction to prevent a breath sample from being escaped therefrom.